The invention relates to improving the potency, absorption or pharmacokinetic properties of therapeutic compounds. The addition of poly(ethylene glycol) or (PEG) is a known method of increasing the half-life of some compounds by reducing kidney clearance, reducing aggregation, and diminishing potentially unwanted immune recognition (Jain, Crit. Rev. Ther. Drug Carrier Syst. 25:403-447 (2008)). The PEG is typically used at a considerably large size (20-40 kDa) to maximize the half-life in circulation. This can be accomplished by using either a single large PEG or multiple smaller PEGs attached to the compound. (Clark et al. J. Biol. Chem. 271:21969-21977 (1996); Fishburn, J. Pharm. Sci. 97:4167-4183 (2008)).
Absorption is a primary focus in drug development and medicinal chemistry since a drug must be absorbed before any medicinal effects can take place. A drug's pharmacokinetic profile can be affected by many factors. Additionally, the absorption properties of therapeutic compounds vary significantly from compound to compound. Some therapeutic compounds are poorly absorbed following oral or dermal administration. Other therapeutic compounds, such as most peptide- and protein-based therapeutics, cannot be administered orally. Alternate routes of administration such as intravenous, subcutaneous, or intramuscular injections are routinely used for some of compounds; however, these routes often result in slow absorption and exposure of the therapeutic compounds to enzymes that can degrade them, thus requiring much higher doses to achieve efficacy.
A number of peptides have been identified as therapeutically promising. The chemical and biological properties of peptides and proteins make them attractive candidates for use as therapeutic compounds. Peptides and proteins are naturally-occurring molecules made up of amino acids and are involved in numerous physiological processes. Peptides and proteins display a high degree of selectivity and potency, and may not suffer from potential adverse drug-drug interactions or other negative side effects. Thus peptides and proteins hold great promise as a highly diverse, highly potent, and highly selective class of therapeutic compounds with low toxicity. Peptides and proteins, however, may have short in vivo half-lives. For such peptides, this may be a few minutes. This may render them generally impractical, in their native form, for therapeutic administration. Additionally, peptides may have a short duration of action or poor bioavailability.
Fibroblast growth factor 21 (SEQ ID:2) is a protein that circulates in serum. Encoded by the FGF21 gene, it is a member of a family of atypical fibroblast growth factors (FGFs), which include FGF19 and FGF23. It lacks the conventional FGF heparin-binding domain. FGF family members possess broad mitogenic and cell survival activities and are involved in a variety of biological processes including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. FGF21 is specifically induced by HMGCS2 activity. FGF21 stimulates glucose uptake in adipocytes but not in other cell types. This effect is additive to the activity of insulin.
In vitro studies indicate that FGF21 prefers binding to the FGFR1c/b-Klotho receptor complex over those containing other FGFR isotypes (Kliewer and Mangelsdorf, Am. J. Clin. Nutr. 91:254S-257S (2010)). FGF21 promotes glucose uptake by adipocytes in vitro. Administration of FGF21 to diabetic animals reduces circulating glucose levels while excess FGF21 does not induce hypoglycemia as seen with administration of excess insulin (Kharitonenkov and Shanafelt, Curr. Opin. Investig. Drugs 10:359-364 (2009)). Therefore, FGF21 is a promising therapeutic protein for the treatment of diabetes. FGF21, however, was administered frequently to see therapeutic benefits in an animal model (Kharitonenkov et al., J. Clin. Invest. 115:1627-1635 (2005)). FGF21 in its natural state has an extremely short half-life in serum (1.1 hr) making exogenous addition of FGF21 in its natural state not clinically practical as a treatment (see WO03/011213). Additionally, FGF21 exhibits poor bioavailability when injected subcutaneously. In a comparative pharmacokinetic study, 1 mg/kg of FGF21 was injected either intravenously (IV) or subcutaneously (SC) and the concentration of FGF21 was analyzed over time. The results showed a significant reduction in bioavailability using a subcutaneous route of administration (Cmax 73 nM) compared to the intravenous route (Cmax of 1890 nM; see Table 1 in Xu J et al., 2009. Am J Physiol Endocrinol Metab 297: E1105-E1114). The foregoing references are incorporated herein by reference in their entirety.
Ghrelin peptide (SEQ ID NO:5) is naturally secreted from the stomach in mammals into circulation to stimulate appetite and release of growth hormone. Ghrelin stimulates the release of growth hormone (GH) from the pituitary gland through the cellular receptor GHS-R and plays important roles in energy homeostasis. In addition, ghrelin acts directly on the central nervous system to decrease sympathetic nerve activity. Ghrelin receptors (GHS-Rs) are concentrated in the hypothalamus-pituitary unit. GHS-R is distributed in peripheral tissues, including the heart, lung, liver, kidney, pancreas, stomach, small and large intestines, adipose, and immune cells.
Ghrelin has been used therapeutically to increase weight and lean body mass in patients suffering from cachexia or involuntary weight loss resulting from a chronic disease such as cancer (Hiura et. al., Cancer Jan. 26, 2012, http://onlinelibrary.wiley.com/doi/10.1002/cncr.27430/abstract). Ghrelin, however, has a naturally short half-life of 11 mins in humans (Akamizu et al., Eur J Endocrinol 150:447-55 (2004)) and thus must be dosed often to see therapeutic effects.
Infliximab (Remicade®, Janssen Biotech Inc., U.S. Pat. No. 5,919,452 and US 2002/0141996, incorporated herein by reference in their entirety) is a monoclonal antibody that binds tumor necrosis factor alpha (TNF-α, SEQ ID:10) that is used to treat autoimmune diseases. Infliximab was approved by the U.S. Food and Drug Administration (FDA) for the treatment of psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, and ulcerative colitis. TNF-α is a chemical messenger (cytokine) and a key part of the autoimmune reaction. Infliximab is administered intravenously by a healthcare professional and is not approved for subcutaneous dosing.